Isolation and activation of porcine natural killer cells

Abstract

Background & Aim Natural killer (NK) cells are major effector cells that are critical to the innate immune system and are typically considered to play a fundamental role, particularly in anti-viral and anti-tumor host responses. NK cells provide rapid responses to virus-infected cells, acting at around 3 days after infection, and respond to tumor formation. Typically, immune cells detect the major histocompatibility complex presented on infected cell surfaces, triggering cytokine release, causing lysis or apoptosis. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus, where they then enter into the circulation. In this study, we isolated, characterized and activated NK cells from porcine peripheral blood mononuclear cells (PBMCs). Methods, Results & Conclusion We firstly peripheral blood mononuclear cells (PBMCs) were isolated from pigs (10 weeks old, n=3). Primary porcine NK cells were isolated from PBMCs by negative MACS depletion of CD3 positive cells followed by FACS purification using antibodies against porcine CD3 (IgG1, clone PPT3) and co-cultured with feeder cells in specific culture condition supplemented with IL-2 (1000 IU/mL), IL-12 (20 ng/mL), IL-15 (20 ng/mL), IL-18 (100 ng/mL) and IL-21 (5 ng/mL) or without supplement for 7 and 14 days. Gamma ray (100 Gy) irradiated K562 cells were used as feeder cells for specific activation of porcine NK cells. As results, porcine CD3 negative cells expressed specific markers of activated NK cells. Therefore, gene levels of activated NK cell cytotoxic receptors (NKp30, NKp44, NKG2D, CD244, perforin, and granzyme B) were significantly higher in culture condition with supplement. These results revealed that porcine NK cells are activated in CD3 negative population when culture condition is supplemented with IL-2, IL-12, IL-15, IL-18 and IL-21.